Altering metabolism in biological processes

ABSTRACT

Compositions of peptides and surface-active agents are described, as are methods of making and using such compositions. The compositions are capable of affecting metabolic rates in biological systems, and to accelerate nutrient uptake without a concomitant increase in biofilm production.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/335,495, filed on Dec. 15, 2008, by Podella, et al. and entitled“ALTERING METABOLISM IN BIOLOGICAL PROCESSES,” now U.S. Pat. No.8,323,949, issued Dec. 4, 2012, which in turn is a continuation of U.S.application Ser. No. 10/799,529, filed on Mar. 11, 2004, by Podella, etal. and entitled “ALTERING METABOLISM IN BIOLOGICAL PROCESSES,” now U.S.Pat. No. 7,476,592, issued Jan. 13, 2009, which in turn claims priorityfrom U.S. Provisional Application Ser. No. 60/454,171, filed Mar. 11,2003, both of which applications are hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates generally to compositions ofsurface-active agents combined with peptides derived from Saccharomycescerevisiae, combinations of which are useful in altering metabolism inbiological processes. The present invention further relates to methodsof making and using compositions comprising surface-active agents incombination with peptides derived from Saccharomyces cerevisiae,particularly for use in treating wastewater.

BACKGROUND OF THE DISCLOSURE

There currently exist methods that can affect the rates by whichbiological metabolic processes proceed. The ability to alter theseprocesses would find applications where reductions in biomass andbiofilm production, accelerated nutrient uptake, improved fermentationrates, as well as methods for altering biochemical processes would bebeneficial.

Acceleration of nutrient uptake without a concomitant increase ofbiomass can be achieved by uncoupling biochemical degradation(catabolism) from biochemical synthesis (anabolism). Uncoupling canoccur during oxidative phosphorylation resulting in lower adenosinetriphosphate (ATP) formation, or by dissipating generated ATP through“energy spilling”. There are some chemical moieties known to uncoupleoxidative phosphorylation, however they are inherently toxic and costprohibitive.

All microorganisms have the common purpose of using catabolism toconserve free energy by distributing it among compounds that can storeand carry energy to where it is required in the cell. Intracellularregulation of catabolic and anabolic processes by bacteria is necessaryto ensure an efficient flow of energy. Atkinson B. and Mavituna F.,Biochemical Engineering and Biotechnology Handbook, 2nd Edition, pp.130-131, Stockton, New York (1991), describe the role of adenosinediphosphate-adenosine triphosphate (ADP-ATP) as follows: The high-energyphosphate bonds of ATP are used in coupled reactions for carrying outenergy-required functions, wherein ultimately ADP and inorganicphosphate are formed. ADP is rephosphorylated to ATP during energyyielding reactions of catabolism. (See FIG. 1). Within the mitochondriaof higher organisms, the concentration of ATP is known to regulate theactivity in the citric acid cycle, in effect producing a feedbackcontrol loop. (Stryer, L., Biochemistry, 3rd Edition. Freeman, New York(1988)).

Senez, J. C., Some Considerations on the Energetics of Bacterial Growth,Bacteriol. Rev. 26, 95-107 (1962), suggests that bacterial anabolism iscoupled to catabolism of substrate through rate-limiting respiration.However, uncoupled metabolism would occur if respiratory control did notexist and instead, the biosynthetic processes were rate limiting.Therefore, the excess free energy would be directed away from theproduction of biomass.

Senez, in describing the link between energy-yielding reactions and theenergy-consuming reaction of cell biosynthesis, conceptualized anyanomaly as “uncoupling”. Russell and Cook (Microbiological Reviews,March 1995, pp. 48-62) declared this all-inclusive definition did notdifferentiate between the production of ATP and the utilization of ATPin non-growth reactions. Because the latter process would be more aptlytermed ATP energy spilling, therefore defining uncoupling as theinability of chemiosmonic mechanisms to generate the theoretical amountof metabolic energy in the form of ATP. This is redefined as “uncoupledoxidative phosphorylation” to differentiate it from other mechanisms.

Stouthamer, A. H., Correlation of Growth Yields In MicrobialBiochemistry, International Review of Biochemistry, Ed. J. R. Quayle,Vol. 21, pp. 1-47, University Park, Baltimore (1979), reports thatuncoupled metabolism has been observed in the presence of the followingconditions: 1) in the presence of inhibitory compounds, 2) in thepresence of excess energy source, 3) at unfavorable temperatures, 4) inminimal media, and 5) during transition periods in which cells areadjusting to changes in their environment.

Low and Chase (Wat. Res., Vol. 33, No. 5, pp. 1119-1132, 1999) theorizethat decreasing the ATP available for biosynthesis would, in turn,reduce biomass production. The ability to replicate these uncouplingprocesses in wastewater treatment would be advantageous. Further, ifmicroorganisms exhibit similar behavior to mitochondria in theregulation of the activity in the citric acid cycle, then a reduction ofcellular ATP production would provide a stimulus to the feedback loop topromote accelerated catabolism of pollutants.

There exist several possibilities for the consumption or loss of energyrequired for biomass production, including the dissipation of energy asheat by adenosine triphosphate systems, the activation of alternativemetabolic pathways bypassing free energy conserving reactions, and theaccumulation of polymerized products in storage form or as secretedwaste.

Protonophores are reagents that exhibit the ability to disrupt the tightcoupling between electron transport and the ATP synthase of therespiratory chain, because they dissipate the proton gradient across theinner mitochondrial membrane created by the electron transport system.Typical examples include: 2,4-dinitrophenol, dicumarol, and carbonylcyanide-p-trifluoromethoxyphenyl hydrazone. These compounds share twocommon features: hydrophobic character and a dissociable proton. Asuncouplers, they function by carrying protons across their innermembrane. Their tendency is to acquire protons on the cytosolic surfaceof the membrane (where the proton concentration is high) and carry themto the matrix side, thereby destroying the proton gradient that coupleselectron transport and the ATP synthase. The energy released in electrontransport is dissipated as heat. Biochemistry, 2nd Edition, Garrett andGrisham (1999)

Addition of protonphores to uncouple the energy generating mechanisms ofoxidative phosphorylation will stimulate the specific substrate rateuptake while reducing the rate of biomass production.

Low and Chase (Wat. Sci. Tech., Vol. 37, No 4-5, pp. 399-402, 1998)supplemented a chemostat monoculture of P. putida with the protonphoricuncoupler of oxidative phosphorylation, para-nitrophenol. The effect ofthis addition was to dissipate energy within the cells and thus reducethe energy available for endothermic processes. Under these conditions,cells continued to satisfy their maintenance energy requirements priorto making energy available for anabolism, thus reducing the observedbiomass yield.

Through catabolism, cells make biologically useful energy available forfueling their endothermic reactions. An increase of the energyrequirements for non-growth activities, in particular maintenancefunctions, would decrease the amount of energy available forbiosynthesis of new biomass. Endothermic maintenance functions includethe turnover of cell materials and osmotic work to maintain gradients.The net effect is a utilization of ATP through ‘futile cycles’ alsocalled energy spilling, which reduces the amount of ATP available forthe synthesis of biomass. Also, energy requirements for cell motilitycannot be differentiated from maintenance energy requirements. (Low andChase, 1998)

The discovery and use of peptides produced from Saccharomyces cerevisiaefor medicinal applications described by George Sperti in U.S. Pat. Nos.2,320,478, 2,320,479 and 2,239,345, illustrate a method for producinglow molecular weight heat-shock proteins, referred to as Live Yeast CellDerivative (LYCD), that has demonstrated the ability to affect skinrespiration. The active peptides in the LYCD have been isolated andidentified by a number of individuals. Bentley, U.S. Pat. No. 5,356,874and Bentley et al. (Peptides From Live Yeast Cell Derivative StimulateWound Healing, Arch Surg, Vol. 125, pp. 641-46, May 1990) describe theactive ingredients as an angiogenic factor comprising a mixture ofpolypeptides having molecular weights ranging between about 6,000daltons to about 17,000 daltons, said factor isolated from a yeast cellderivative. Schlemm et al. (Medicinal Yeast Extracts, Cell Stress &Chaperones (1999) 4(3) 171-76) further defines these extracts as the15,700 dalton yeast copper, zinc-superoxide dismutase, the 10,100 daltonacyl CoA binding protein (ACBP), the 8,560 dalton ubiquitin protein andthe 7,090 dalton peptide of the C-terminal fragment of heat-shockprotein 12, a glucose-lipid regulated protein.

Enzymatic compositions founded on yeast extracts have been used for thetreatment of wastewater. Battistoni, U.S. Pat. No. 3,635,797, describesa multi-enzymatic composition comprised of an enzymatic fermentationreaction product, surfactants, citric and lactic acids, urea and pineoil. Battistoni claims the composition greatly improves sewage treatmentfacility capabilities by stimulating bacterial growth, eliminatingodors, and enzymatically improving the catalytic degradation of sewageimpurities. The Battistoni patent describes an anaerobic yeastfermentation process as follows:

“Approximately 1,000 gallons of warm softened water having a temperatureof between about 85-100 degrees F. was placed in a large tank. To thewater was added 700 pounds of black untreated cane molasses, 210 poundsraw cane sugar and 10 pounds magnesium sulfate. The mixture wasthoroughly blended, after which 95 pounds diastatic malt and 10 poundsbakers yeast were added and agitated slightly. The composition wasallowed to stand for about 3 days, after which the effervescent reactionhad subsided, indicating essentially complete fermentation.”

Because it is performed without active aeration or agitation, thefermentation process described in Battistoni is considered anaerobic innature.

Dale, U.S. Pat. No. 5,879,928, describes a composition for the treatmentof municipal and industrial wastewater, comprised of a yeastfermentation supernatant, preservatives and a non-ionic surfactant. Thecomposition comprises a fermentation supernatant from a Saccharomycescerevisiae culture, sodium benzoate, imidazolidinyl urea, diazoldinylurea and a non-ionic surfactant. The Dale patent describes a compositionhaving desirable properties associated with surfactant micro bubbles.Dale explains that the micro bubbles formed with the composition appearto increase the mass transfer of oxygen in liquids. Further, the microbubbles are the result of aggregates of surfactant molecules with aloose molecular packing more favorable to gas mass transfercharacteristics because a surface consisting of fewer molecules would bemore gas permeable than a well-organized micelle containing gas. Dalefurther describes biologically derived catalysts in combination with thesurfactants, both of which tend to be amphiphilic; that is, they havepronounced hydrophobic and hydrophilic properties. The non-ionicsurfactants used in the Dale composition are said to be compatible with,and enhance enzymatic reactions. However, Dale also states thecomposition has catalytic activities that are more like the catalyticactivities of functionalized surfactants than conventional enzymesystems.

The composition of the Dale patent is similar to that described in theBattistoni patent; that is, the fermentation process is anaerobic innature, with an added step of the removal of the resulting yeast cellsby centrifugation. The Dale fermentation process is described asfollows:

“The yeast, Saccharomyces cerevisiae, is cultured in a mediumcomprising: a sugar source, such as sucrose from molasses, raw sugar,soy beans or mixtures thereof. A sugar concentration of about 10 toabout 30%, by weight; malt, such as diastatic malt at a concentration ofabout 7 to about 12%, by weight; a salt, such as magnesium salts, and inparticular magnesium sulfate, at a concentration of about 1 to about 3%,by weight, and yeast is added to the medium to a final concentration onabout 1 to about 5%, by weight, is used. The mixture is incubated atabout 26 degrees to about 42 degrees C. until the fermentation iscompleted, i.e. until the effervescence of the mixture has ceased,usually about 2 to about 5 days, depending on the fermentationtemperature. At the end of the fermentation, the yeast fermentationcomposition is centrifuged to remove the “sludge” formed during thefermentation.”

Consistent with the Battistoni patent, without the presence of activeaeration or agitation, the fermentation process described in the Dalepatent is considered anaerobic in nature. Carbon mass balance studies,conducted under controlled conditions, indicate the Battistonicomposition increases the rate of carbon metabolism versus an untreatedcontrol. Likewise, carbon mass balance studies conducted with the Dalecomposition yielded an even greater increase in the rate of carbonmetabolized versus Battistoni and the control. However, the rate ofconversion of carbon metabolized to biomass carbon of either compositionremained relatively consistent with the conversion rate of the untreatedcontrol. This would indicate the uncoupling of metabolism is not afunction of either the Dale or Battistoni compositions. Nor are the lowmolecular weight peptides described in the present application producedas a result of the anaerobic fermentation of Saccharomyces cerevisiae asdescribed in either of the Battistoni or Dale patents.

Production of excess biomass during biological treatment of wastewatersrequires costly disposal. With environmental and legislative constraintslimiting disposal options, considerable impetus exists for reducing theamount of biomass produced. (Low E. W. and Chase H. A., The Effect ofMaintenance Energy Requirements on Biomass Production During WastewaterTreatment, Water Research, Vol. 33, Issue 3, pp. 847-853. (2000)). Theactivated sludge process employs a microbial population that willconvert organic pollutants to cell mass and respiration products. Cellmass accumulates within the process and the excess biomass musttherefore be disposed of. Although such treatment and disposal mayalready account for 60% of total plant operating costs, (Horan N. J.,Biological Wastewater Treatment Systems, Wiley, Chichester (1990)),these costs are expected to rise with new European Community (EC)legislation and decreasing landfill availability. Sludge disposal in theUnited States has also come under increasing scrutiny and newlegislation regulating sludge disposal is being enacted by governmentagencies at all levels. (Low and Chase, The Use of Chemical Uncouplersfor Reducing Biomass Production During Biodegradation, Wat. Sci. Tech.,Vol. 37, No. 4-5, pp. 399-402, 1998).

Dissipating energy intended for anabolism of cell mass without reducingthe rate of removal of organics from aqueous waste provides a directmechanism for reducing the yield of biomass. The chemiosmonic mechanismof oxidative phosphorylation (by which adenosine diphosphate (ADP) toenergy-rich adenosine triphosphate (ATP) is produced during catabolism,(Mitchell, P., Chemiosmonic Coupling and Energy Transduction: A LogicalDevelopment of Biochemical Knowledge, Bioenergetics 3, 5-24 (1961)), canbe uncoupled using protonphores and under these circumstances isdissipated. Oxidation of the substrate still occurs, but thephosphorylation of ADP to ATP is reduced, and consequently, there isless energy available for the formation of biomass. (Simon, E. W.,Mechanisms of Dinitrophenol Toxicity, Biol. Rev. 28, 453-479 (1953)).

SUMMARY OF THE INVENTION

Investigation into the use of yeast fermentation by-products for thepurpose of ascertaining the degree to which these compounds affectbiochemical metabolic shifts have resulted in the discovery of a groupof low molecular weight proteins that, when combined with surface-activeagents, result in an increased rate of catabolism without a concomitantincrease in biomass production. While these low molecular weightproteins can be produced by Saccharomyces cerevisiae during aerobicfermentation as practiced by those familiar in the art, their yieldincreases significantly if the yeast cells are placed under stressconditions during or near the end of the fermentation process. Thesestress conditions can occur during periods of very low food to massconcentrations, or as the result of heat shock or pH shock conditions asdescribed in U.S. Pat. No. 6,033,875, Bussineau et al.

It has further been found that compositions of Live Yeast CellDerivative—which is an alcoholic derivative from Saccharomycescerevisiae produced by the methods set forth U.S. Pat. Nos. 2,239,345,2,230,478 and 2,230,479—coupled with surface-active agents, produceeffects that simulate uncoupling of biochemical metabolic pathways whenadded to mixed-culture aerobic processes. The crude Live Yeast CellDerivative was further refined utilizing dialysis membranes as set forthin U.S. Pat. No. 5,356,874, Bentley, yielding polypeptides having themolecular weights ranging between 6,000 and 17,000 daltons as determinedby SDS-page electrophoresis. (Bentley, et al., 1990).

These low molecular weight (6-17 kD) proteins, in combination withsurface-active agents, were found to yield an increase in catabolism oforganic matter without the proportional increase in biomass, and also toincrease the amount of carbon dioxide respired. Studies demonstrated theuse of either the low molecular weight proteins or the surface-activeagents alone showed little effect on the catabolic or anabolic rates. Asynergistic effect was observed when surface-active agents were combinedwith the low molecular weight proteins of the present invention.

Thus, it is an object of this invention to describe compositions, basedon yeast-derived low molecular weight proteins and surface-activeagents, which have the ability to alter biological metabolic processesin such a way as to simulate the uncoupling of biochemical pathways.

It is another object of this invention to describe methods by which theproduction of low molecular weight proteins from Saccharomycescerevisiae may be enhanced.

It is a further object of this invention to utilize the describedcompositions and methods to affect the catabolic and anabolic biologicalprocesses during the treatment of municipal and industrial wastewatertreatment processes for the purposes of accelerating the biologicaldegradation of organic wastewater contaminants, reducing the productionof activated sludge, reducing the amount of aeration required as aresult of the accelerated catabolic processes, reduction of biofilm incross-flow membrane filtration and cooling towers, and cleaningcompositions in non-sterile environments.

It is yet another object of the invention to stabilize the compositionsthrough the use of anti-microbial agents and pH adjustment to providelong-term stability to the compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of the role of the ATP-ADP cycle incell metabolism. (Adapted from B. Atkinson and F. Mavituna, BiochemicalEngineering and Biotechnology Handbook, 2nd Edition, pp. 130-131,Stockton, New York (1991)).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the specification, the following terms are defined as follows:

The “aerobic yeast fermentation process” is defined as the standardpropagation conditions utilized in the production of commerciallyavailable baker's yeast as described in the chapter entitled “Baker'sYeast Production”, in Nagodawithana T. W. and Reed G., NutritionalRequirements of Commercially Important Microorganisms, EsteekayAssociates, Milwaukee, Wis., pp 90-112 (1998), and further describedbelow.

“Live Yeast Cell Derivative (LYCD)” is defined as an alcoholic extractobtained from baker's yeast prepared as described below.

The “uncoupling factors” are defined as the biologically activepolypeptide fraction obtained from LYCD. While the preferred method forobtaining LYCD is an alcoholic extract, and the peptides of the“uncoupling factors” are found in the LYCD, we show that these“uncoupling factors” need not be derived from an alcoholic extract, butcan be generated from an aerobic fermentation, and preferably followedby a heat shock process and some form of cell disruption.

The “surface-active agents” are defined as non-ionic and anionicsurfactants as described below.

The present inventors have isolated an uncoupling factor derived fromLYCD of aerobic yeast fermentation processes which, when coupled withsurface-active agents, simulates uncoupling oxidative phosphorylation orenergy spilling in biochemical processes. This factor is comprised ofpolypeptide fractions ranging in molecular weights between about 6,000and 17,000 daltons as indicated by results of polyacrylamide gelelectrophoresis. The polypeptide uncoupling factor of the presentinvention, when utilized in isolation, appears to have little or noeffect on biological metabolism. Likewise, the use of the surface-activeagents of the present invention in isolation, does not lead to asignificant change of the metabolic rate. There is a synergistic effectcreated through the combination of surface-active agents with theuncoupling factor derived from yeast, that affect a profound change ofthe metabolism by uncoupling biochemical pathways.

The uncoupling factor may be produced by an aerobic yeast fermentationprocess. Yeast fermentation processes are generally known to those ofskill in the art, and are described, for example, in the chapterentitled “Baker's Yeast Production” in Nagodawithana T. W. and Reed G.,Nutritional Requirements of Commercially Important Microorganisms,Esteekay Associates, Milwaukee, Wis., pp 90-112 (1998), which is herebyincorporated by reference. Briefly, the aerobic yeast fermentationprocess is conducted within a reactor having aeration and agitationmechanisms, such as aeration tubes and/or mechanical agitators. Thestarting materials (liquid growth medium, yeast, sugars, additives) areadded to the fermentation reactor and the fermentation is conducted as abatch process.

After fermentation, the fermentation product may be subjected toadditional procedures intended to increase the yield of uncouplingfactor produced from the process. Several examples of post-fermentationprocedures are described in more detail below. Other processes forincreasing yield of uncoupling factor from the fermentation process maybe recognized by those of ordinary skill in the art.

Although saccharomyces cerevisiae is a preferred starting material,several other yeast strains may be useful to produce yeast fermentmaterials used in the compositions described herein. Additional yeaststrains that may be used instead of or in addition to Saccharomycescerevisiae include Kluyveromyces marxianus, Kluyveromyces lactis,Candida utilis (Torula yeast), Zygosaccharomyces, Pichia, Hansanula, andothers known to those skilled in the art.

The surface-active agents used in the compositions are preferablysurfactants. Surfactants that are useful in the compositions describedherein may be either nonionic, anionic, amphoteric or cationic, or acombination of any of the above, depending on the application. Suitablenonionic surfactants include alkanolamides, amine oxides, blockpolymers, ethoxylated primary and secondary alcohols, ethoxylatedalkylphenols, ethoxylated fatty esters, sorbitan derivatives, glycerolesters, propoxylated and ethoxylated fatty acids, alcohols, and alkylphenols, alkyl glucoside glycol esters, polymeric polysaccharides,sulfates and sulfonates of ethoxylated alkylphenols, and polymericsurfactants. Suitable anionic surfactants include ethoxylated aminesand/or amides, sulfosuccinates and derivatives, sulfates of ethoxylatedalcohols, sulfates of alcohols, sulfonates and sulfonic acidderivatives, phosphate esters, and polymeric surfactants. Suitableamphoteric surfactants include betaine derivatives. Suitable cationicsurfactants include amine surfactants. Those skilled in the art willrecognize that other and further surfactants are potentially useful inthe compositions depending on the particular application. For example, ablend of non-ionic and anionic surfactants has been found to provideparticularly good results.

Preferred anionic surfactants used in the composition include CalFoam™ES 603, a sodium alcohol ether sulfate surfactant manufactured by PilotChemicals Co., and Steol™ CS 460, a sodium salt of an alkyl ethersulfate manufactured by Stepan Company. Preferred nonionic surfactantsused in the enzyme/surfactant compound include Neodol™ 25-7 or Neodol™25-9, which are C12-C15 linear primary alcohol ethoxylates manufacturedby Shell Chemical Co., and Genapol™ 26 L-60, which is a C12-C16 naturallinear alcohol ethoxylated to 60E C cloud point (approx. 7.3 mol),manufactured by Hoechst Celanese Corp. It should be understood thatthese surfactants and the surfactant classes described above areidentified only as preferred materials and that this list is neitherexclusive nor limiting of the composition.

Turning to the preferred embodiments, the compositions comprise anaerobic yeast fermentation supernatant combined with surface-activeagents. In the preferred embodiment, saccharomyces cerevisiae is grownunder aerobic conditions familiar to those skilled in the art, using asugar, preferably molasses as the primary nutrient source. Additionalnutrients may include, but are not limited to, diastatic malt,diammonium phosphate, magnesium sulfate, ammonium sulfate zinc sulfate,and ammonia. The yeast is preferably propagated under continuousaeration and agitation between 30 degrees to 35 degrees C. and at a pHof 4.0 to 6.0. The process takes between 10 and 25 hours and ends whenthe fermentation broth contains between 4 and 8% dry yeast solids,(alternative fermentation procedures may yield up to 15-16% yeastsolids), which are then subjected to low food-to-mass stress conditionsfor 2 to 24 hours. Afterward, the yeast fermentation product iscentrifuged to remove the cells and cell fragments, and thesurface-active agents are added to the supernatant.

In an alternative embodiment, the yeast fermentation process is allowedto proceed until the desired level of yeast has been produced. Prior tocentrifugation, the yeast in the fermentation product is subjected toheat-stress conditions by increasing the heat to between 40 and 60degrees C., for 2 to 24 hours, followed by cooling to less than 25degrees C. The yeast fermentation product is then centrifuged to removethe yeast cell debris and the supernatant is blended with surface-activeagents.

In a further alternative embodiment, the fermentation process is allowedto proceed until the desired level of yeast has been produced. Prior tocentrifugation, the yeast in the fermentation product is subjected tophysical disruption of the yeast cell walls through the use of a FrenchPress, ball mill, high-pressure homogenization, or other mechanical orchemical means familiar to those skilled in the art, to aid the releaseof intracellular, low molecular weight polypeptides. It is preferable toconduct the cell disruption process following a heat shock, pH shock, orautolysis stage. The fermentation product is then centrifuged to removethe yeast cell debris and blended with surface-active agents.

In a still further alternative embodiment, the fermentation process isallowed to proceed until the desired level of yeast has been produced.Following the fermentation process, the yeast cells are separated out bycentrifugation. The yeast cells are then partially lysed by adding 2.5%to 10% of the surfactant used in the final product formulation to theseparated yeast cell suspension (10%-20% solids). In order to diminishthe protease activity in the yeast cells, 1 mM EDTA is added to themixture. The cell suspension and surfactants are gently agitated at atemperature of about 25° to about 35° C. for approximately one hour tocause partial lysis of the yeast cells. Cell lysis leads to an increasedrelease of intracellular small proteins. After the partial lysis, thepartially lysed cell suspension is blended back into the ferment andcellular solids are again removed by centrifugation. The product is thenblended with surface-active agents.

In a still further alternative embodiment, fresh live Saccharomycescerevisiae is added to a jacketed reaction vessel containingmethanol-denatured alcohol. The mixture is gently agitated and heatedfor two hours at 60 degrees C. The hot slurry is filtered and thefiltrate is treated with charcoal and stirred for 1 hour at ambienttemperature, and filtered. The alcohol is removed under vacuum and thefiltrate is further concentrated to yield an aqueous solution containingthe heat shock proteins. This LYCD composition is then blended withwater and surface-active agents.

In a still further alternative embodiment, the LYCD is further refinedso as to isolate the active uncoupling factors utilizing Anion ExchangeChromatography of the crude LYCD, followed by Molecular SieveChromatography. The refined LYCD is then blended with water andsurface-active agents.

In a still further alternative embodiment, the surface-active agents ofthe compositions are comprised of non-ionic surfactants.

In a still further alternative embodiment, the surface-active agents ofthe compositions are comprised of a blend of non-ionic surfactants andanionic surfactants.

In a still further alternative embodiment, preservatives and stabilizersare added to the compositions and the pH is adjusted to between 3.8 and4.8 to provide long-term stability to the composition.

In a still further alternative embodiment, the compositions are used inthe treatment of municipal and industrial wastewater wherein degradationof biologically available wastewater contaminants is accelerated withouta concomitant increase in the production of sludge.

The compositions prepared by the preceding processes are useful in anumber of applications where bacteria, or yeast, are utilized in theapplication. For example, the compositions may be useful in applicationshaving as a goal the destruction or removal of organic compounds, suchas treating wastewater or other bioremediation. Alternatively, thecompositions may be useful in applications having as a goal the creationof fermentation beer or antibiotics. Other types of applications forwhich the compositions may be useful are those in which biomass orbiofilm production is sought to be minimized, which is achieved by thecompositions described herein by the uncoupling of the ATP cycle and theeffect on reproduction of bacteria and production of polysaccharides(biofilm). These applications are useful, for example, in the reductionof hydrogen sulfide in sewers where bacteria form biofilm, thus creatinganaerobic conditions that are conducive to the production of hydrogensulfide, or in the reduction of calcium carbonate in cooling towerswhere calcium salts peg onto biofilm and clog circulation systems, or inthe minimization or elimination of biofilm in medical products. Specificapplications include, but are not limited to, the following: municipalwastewater plants, industrial wastewater plants (particularly foodprocessing), sewage lines, septic tanks and septic fields, cross-flowmembrane filtration systems, cooling towers, soil remediation,bilgewater in ships, odor control applications, agriculturalapplications (e.g., cattle and chicken manure ponds), clarification ofstanding bodies of water in facultative lakes, ponds, and lagoons,pools/spas, cleaners for non-sterile environments, and the like.Although several examples are listed, the person of ordinary skill inthe art will appreciate that the compositions described herein will haveadditional applications not explicitly set forth herein, but which arenatural extensions of those listed and/or which are contemplated by thegeneral mode of action of these compositions as described herein.

Test Method

The carbon mass balance studies described herein utilized a sterilenutrient broth solution using Bacto Tryptic Soy Broth, produced byBecton Dickinson and Company, that was inoculated with Polyseed, aproprietary blend of aerobic bacteria produced by InterLab, approved bythe U.S. Environmental Protection Agency for Biological Oxygen Demand(BOD) determination. Tryptic Soy Broth was chosen as a nutrient becauseit is a defined Chemical Composition and is completely soluble. Hence,any suspended solids or particulate matter that develop during thecourse of the study is assumed to be biomass produced as a result of theassimilation of organic carbon. Since it is known that approximately 51%of a bacterial cell consists of carbon, one can determine the rate ofcarbon in the nutrient substrate that is converted to biomass byanalyzing unfiltered versus filtered samples for total organic carbon atthe beginning of the experiment, followed by sample analysis at anypoint in time during the study.

Carbon mass balance studies were conducted to determine the ability ofthe present invention to affect changes in carbon uptake, rate ofconversion of carbon to biomass or to carbon dioxide, respectively. Thestudies were conducted utilizing a 2 liter reactor vessel, Applikon BioConsole, Model ADI 1025 and an Applikon BioController, Model ADI 1010using air that had been sparged through a 1.5N sodium hydroxide solutionfollowed by sparging through 2× deionized water to remove all carbondioxide from the aeration source. The bioreactor exhaust air was thensparged through a 1.5N sodium hydroxide solution trap and the amount ofcarbon dioxide respired in the bioreactor during the test period wasdetermined.

A Tryptic Soy Broth solution was prepared by adding 72 grams of sterile10% Tryptic Soy Broth concentrate to 2400 ml of 2×-deionized water in a4 l. beaker. Two capsules of Polyseed inoculum were added to thenutrient solution. The inoculated nutrient was warmed up and maintainedat 30 degrees C., with continuous agitation using a stir bar for 14hours. Prior to transferring the nutrient to the bioreactor, thenutrient solution was filtered through 4 layers of cheesecloth to removethe grain used as the substrate for the bacteria inoculum. Two liters ofthe nutrient solution were charged into the bioreactor. The “Treated”samples had 10 mg/l of the test composition (unless different doselevels are noted) added to the nutrient, while deionized water was addedto the “Control” at the same 10 mg/l.

The bioreactor was then sealed and carbon dioxide-free air was spargedat a feed rate of 1.0 liter per minute while the bioreactor temperaturewas maintained at 30 degrees C. and the turbine mixer ran at 500 RPM forthe duration of the test. The exhaust air was sparged through a 1.5 Msodium hydroxide solution to capture the carbon dioxide being respired.

Upon completion of the test, the sodium hydroxide solution wastransferred to a beaker and 20 ml of a 3.5N barium chloride solution wasadded. The solution was neutralized with 4N hydrochloric acid using a pHmeter and a buret to determine the volume of hydrochloric acid solutionrequired to neutralize the solution. This is the value for B. Thestandardization factor was created by neutralizing 200 ml of 1.5N sodiumhydroxide solution with 4N hydrochloric acid using a pH meter and aburet to determine the volume of hydrochloric acid solution required toneutralize the solution. This determined the value for S. The amount ofcarbon respired as carbon dioxide was then calculated using S and B inthe following equation: C=6N(B−S) where N=7.5

The nutrient was sampled at 0 hours and again at the conclusion of thestudy. Filtered and unfiltered nutrient samples were analyzed for totalorganic carbon (TOC) using a Shimadzu Total Organic Carbon Analyzer,Model TOC-5000A.

The Carbon Mass Balance is calculated as follows: Carbon NutrientConsumed=Carbon Biomass Increase+Carbon Respired as Carbon Dioxide.

Determination of the level of biomass carbon was achieved by determiningthe difference between the total TOC and soluble TOC of any sample.

Evaluations of Compositions

The compositions described herein have utility in the treatment ofmunicipal and industrial wastewater by accelerating the metabolism ofbiologically available nutrients without producing a concomitantincrease biomass. This feature creates opportunities for the operatorsof these facilities to achieve greater wastewater throughput, relieveovercapacity conditions, and reduce the expense required for sludgedisposal. Tables 2, 4, 6, 8, 10, 12, 14 and 15 demonstrate the abilityof the compositions to achieve these benefits.

EXAMPLE 1

Saccharomyces cerevisiae was cultivated under aerobic conditionsfamiliar to those skilled in the art, using molasses as the primarynutrient source. Additional nutrients can include diastatic malt,diammonium phosphate, magnesium sulfate, ammonium sulfate zinc sulfate,and ammonia. The yeast was propagated under continuous aeration andagitation between 30 degrees and 35 degrees C. and a pH range of between4.0 and 6.0 until the yeast attained a minimum level of 4% based on dryweight and the yeast was then subjected to low food-to-mass stressconditions for a period of 4 hours. At the conclusion of thefermentation process, the yeast fermentation product was centrifuged toremove the yeast cells and the supernatant was then blended withsurface-active agents and the pH adjusted between 3.8 and 4.8 forstability.

EXAMPLE 2

Saccharomyces cerevisiae was cultivated utilizing the conditions foundin Example 1. At the conclusion of the fermentation process, thefermentation product was heated to 50 degrees C. for eight hours so asto induce the heat shock response and perform autolysis on the yeastcells, thus releasing the heat-shock proteins into the growth media. Atthe conclusion of the autolysis process, the yeast fermentation productwas centrifuged to remove the yeast cells and the supernatant was thenblended with surface-active agents and the pH adjusted between 3.8 and4.8 for stability.

EXAMPLE 3

Saccharomyces cerevisiae was cultivated utilizing the conditions foundin Example 1. At the conclusion of the fermentation process, thefermentation product was heated to 40 degrees C. for two hours so as toinduce the heat shock response and perform autolysis on the yeast cells,thus releasing the heat-shock proteins into the growth media. At theconclusion of the autolysis process, the fermentation product was passedthrough a Manton-Gaulin High Pressure Homogenizer at 7000 psi for threecycles, after which the yeast fermentation product was centrifuged toremove the yeast cell debris and the supernatant was then blended withsurface-active agents and the pH adjusted between 4.0 and 4.6 forstability. Formulae for the three examples are as follows:

TABLE 1 % By Weight Material Example 1 Example 2 Example 3 Non-autolysedFerment 64.50% Autolysed Ferment 64.50% Disrupted Cell Ferment 64.50%Ethoxylated Linear Alcohol - 7 mole 26.60% 26.60% 26.60% ETO Alkyl EtherSulfate, Sodium Salt 8.90% 8.90% 8.90% (60%) Adjust pH to 4.0 withPhosphoric Acid Total 100.00% 100.00% 100.00%

As shown in Table 2, results of the carbon mass balance bench test usingExamples 1-3 at 10 mg/l versus an untreated control indicate an increasein carbon uptake of between 100-160% when compared to the control,without a concomitant increase in biomass. Respiration rates exhibited a4 to 5 fold increase for the treated samples. Results were as follows:

TABLE 2 Carbon Mass Balance Comparisons of Modified AerobicFermentations Processes vs.: Control Control Example 1 Example 2 Example3 Nutrient TOC/mg/l 0 Hour 891.2 924.3 863.6 854.0 4 Hours 715.9 571.3509.1 398.1 ΔTOC 175.3 353.0 430.0 455.9 Biomass TOC/mg/l 0 Hour 81.265.9 39.2 71.7 4 Hours 224.0 213.1 277.5 321.9 ΔTOC 1 142.8 147.2 238.3250.2 Nutrient TOC to Biomass 81.5 41.7 55.5 55.3 TOC % Carbon Dioxideas Carbon 49.5 198.0 220.5 252.0 mg/l

EXAMPLE 4

Composition is based on U.S. Pat. No. 3,635,797 wherein the fermentationprocess was conducted under anaerobic conditions; that is, no activeaeration was applied to the fermentation vessel during the fermentationprocess, and no agitation was utilized. The ferment was allowed to standfor about 3 days, after which the effervescent reaction had subsided.

EXAMPLE 5

Composition is based on U.S. Pat. No. 5,879,928 wherein the fermentationprocess was conducted under anaerobic conditions wherein thefermentation mixture is incubated at about 26 degrees to about 42degrees C. until the fermentation is completed, i.e. until effervescenceof the mixture has ceased, usually about 2 to about 5 days, depending onthe fermentation temperature. At the end of the fermentation, the yeastfermentation composition is centrifuged to remove the “sludge” formedduring the fermentation.

TABLE 3 % By Weight Material Example 4 Example 5 FermentationSupernatant 24.96% 20.00% Water 61.76% 70.54% Nonionic Surfactants 1.89%Anionic Surfactants 4.53% 9.00% Inorganic Salts 1.20% Lactic Acid 0.93%Citric Acid 0.17% Urea 4.20% Pine Oil 0.36% Sodium Benzoate 0.30%Imidazolidinyl Urea 0.01% Diazolidinyl Urea 0.15% Total 100.00% 100.00%

Results of the carbon mass balance studies compare the fermentationproduct differences between anaerobic processes of the prior art and theaerobic process of the present invention. Example 4, utilizing theteachings of U.S. Pat. No. 3,635,797 and treated with the composition ata dose rate of 30 mg/liter, affected a 40.2% increase in nutrient carbonuptake during the 4 hour duration of the study, and also a 38.4%increase in biomass production. However, the rate of nutrient carbonconverted to biomass carbon remained virtually identical, 80.4%conversion for the Battistoni composition vs. 81.5% for the control.

Example 5, utilizing the teachings of U.S. Pat. No. 5,879,928 and alsotreatment level of the composition of 30 mg/liter, affected a 107.8%increase in carbon uptake vs. the

control and 48.3% increase vs. the Battistoni composition, but also135.4% and 128.4% increased in biomass production vs. the control andBattistoni composition, respectively. The amount of nutrient carbonbeing converted to biomass carbon increased over that of the control andExample 4 to a 92.3% carbon conversion rate.

Example 1, utilizing an aerobic fermentation procedure with a treatmentlevel of 10 mg/liter, experienced a significant reduction in theconversion of nutrient carbon to biomass carbon, exhibiting at 48.8%reduction in biomass production relative to the amount of carbonmetabolized. Moreover, the overall metabolism of nutrient carbon wasessentially equivalent (3.2% decline) compared to the Dale compositionand superior to that of Battistoni (43.7% increase) and the control(101.4% increase).

Examination of the protein concentrations of the products produced inthe foregoing examples revealed the following:

-   -   Example 1 protein concentration: 33.38 mg/ml    -   Example 4 protein concentration: 13.88 mg/ml    -   Example 5 protein concentration: 3.01 mg/ml        Thus, the anaerobic processes described in the Battistoni (U.S.        Pat. No. 3,635,797) and Dale (U.S. Pat. No. 5,879,928) patents        produced fermentation products having a significantly lower        protein concentration than the fermentation products produced by        the aerobic processes described herein.

Examination of the amount of nutrient carbon converted to biomasscarbon, as well as the rates of carbon metabolism for the aboveexamples, demonstrate the presence of an uncoupling factor when anaerobic fermentation process is implemented vis-à-vis the anaerobicprocesses described in the prior art. Whereas the compositions of thepresent invention exhibited a significant increase in the metabolism ofthe nutrient without a concomitant increase in biomass, compositions ofthe prior art demonstrated an ability to accelerate nutrient uptake,however the increase of biomass production was commensurate with theamount of nutrient assimilated. Results of this study were as follows:

TABLE 4 Carbon Mass Balance Comparisons of Fermentation Intermediates ofU.S. Pat. No. 3,635,797, U.S. Pat. No. 5,879,928 and Example 1 vs.Control Control Example 1 Example 4 Example 5 Nutrient TOC/mg/l 0 Hour891.2 924.3 897.6 873.4 4 Hours 715.9 571.3 651.9 509.1 ΔTOC 175.3 353.0245.7 364.3 Biomass TOC/mg/l 0 Hour 81.2 65.9 48.1 70.8 4 Hours 224.0213.1 245.7 407.0 ΔTOC 142.8 147.2 197.6 336.2 % Nutrient TOC 81.5 41.780.4 92.3 to Biomass TOC CO2 as 49.5 198.0 144.0 144.0 Carbon mg/l

EXAMPLE 6

The formula was prepared in the same manner as shown in Example 3,except the surfactants were replaced by water to demonstrate thepreference for inclusion of surface-active agents.

EXAMPLE 7

The formula was prepared using the surfactant combination utilized inExample 3 with water replacing the ferment so as to demonstrate theminimal effect of surface-active agents on the biological metabolicprocesses.

TABLE 5 % By Weight Material Example 6 Example 7 Autolysed Ferment64.50% Water 35.50% 64.50% Ethoxylated Linear Alcohol - 7 mole ETO26.60% Alkyl Ether Sulfate, Sodium Salt (60%) 8.90% Adjust pH to 4.0with Phosphoric Acid Total 100.00% 100.00%

Results of the mass balance bench tests shown in Table 6 indicate nosignificant shift in carbon uptake when applying fermentation productalone as in Example 6 at 10 mg/l or with surface-active agents as inExample 7 at 10 mg/l when compared to the untreated control. Resultswere as follows:

TABLE 6 Carbon Uptake Comparisons of Modified Aerobic FermentationsProcesses - With and Without Surfactants, Surfactants WithoutFermentation Product vs. Control Control Example 1 Example 6 Example 7Nutrient TOC/mg/l 0 Hour 894.1 924.3 910.7 901.3 4 Hours 705.0 571.3729.9 737.5 ΔTOC 189.1 353.0 180.8 163.8 Reduction: 21.2% 38.2% 19.9%18.2%

Utilization of nonionic and anionic surfactants at varyingconcentration, and their relationship to ferment at varyingconcentrations, impact the rate at which catabolism proceeds. A protocolwas developed to determine these differences utilizing Tryptic Soy Brothat 3000 mg/liter as a carbon source, inoculated with aerobic bacteria,and incubated for 14 hours. The inoculated nutrient was stirred toensure equal distribution of bacteria, and divided into 1 litergraduated cylinders. One cylinder was not treated for a control and theremaining cylinders were treated with compositions from Examples 8-13 ata dose level of 30 mg/L. All Examples utilized Disrupted Cell Ferment.

EXAMPLE 8

A combination of nonionic and anionic surfactants were utilized at atotal active level of 9%, a disrupted cell ferment level of 20%, withwater making up the balance of the composition.

EXAMPLE 9

Composition replicated Example 8, except the total surfactant level wasreduced by 50%, however the ratio of nonionic to anionic surfactants wasretained.

EXAMPLE 10

Composition replicated Example 9, except the level of disrupted cellferment was increased by 50% and the water concentration was reduced by10%.

EXAMPLE 11

Composition replicated Example 8, except the anionic surfactant wasreplaced by water in the composition.

EXAMPLE 12

Composition replicated Example 8, except the nonionic surfactant wasreplaced by water in the composition.

EXAMPLE 13

Composition replicated Example 10, except both anionic and nonionicsurfactants were replaced with water in the composition.

Abbreviations: ELA Ethoxylated Linear Alcohol - 7 mole ETO AES AlkylEther Sulfate, Sodium Salt (60%) WAT Water DCF Disrupted Cell Ferment

TABLE 7 % By Weight Material EX. 8 EX. 9 EX. 10 EX. 11 EX. 12 EX. 13 DCF20.00% 20.00% 30.00% 20.00% 20.00% 30.00% ELA 7.50% 3.75% 3.75% 7.50%AES 2.50% 1.25% 1.25% 2.50% WAT 70.00% 75.00% 65.00% 72.50% 77.50%70.00% Adjust pH to 4.0 with Phosphoric Acid TOTAL 100.00% 100.00%100.00% 100.00% 100.00% 100.00%

Results demonstrated the interaction of the anionic surfactant whencoupled with a nonionic surfactant and the disrupted cell ferment.Reducing the surfactant combination in Example 9 by 50% of the levelutilized in Example 8 exhibited an 8% reduction in nutrient beingmetabolized. Increasing the ferment portion of the composition by 50% inExample 10, while maintaining the reduced surfactant level, resulted ina 20% increase in carbon metabolized when compared to Examples 8 and 9.Example 11, which contained only the nonionic surfactant and disruptedcell ferment product exhibited an 18% increase in efficacy, versusExhibit 12, containing only the anionic surfactant with the fermentshowed a slight decline. When the disrupted cell ferment was utilized atthe same level as utilized in Exhibit 10, without benefit of anysurfactant, a 75% decline in efficacy resulted for Example 13 versus theExhibit 10 composition, and a 35% reduction when compared to theuntreated control. Results were as follows:

TABLE 8 Relationships Between Nonionic Surfactants, Anionic Surfactantsand Disrupted Cell Fermentation Product Compositions and Their Effectson Metabolic Rates Nutrient TOC = mg/L Time Cont Ex. 8 Ex. 9 Ex. 10 Ex.11 Ex. 12 Ex. 13 Hour 0 920.0 920.0 920.0 920.0 920.0 920.0 920.0 2859.3 697.4 758.1 597.1 533.6 747.0 857.4 4 782.0 560.3 644.9 469.2458.2 579.6 826.2 6 728.6 511.5 543.7 427.8 437.0 528.1 796.7 ΔTOC 191.4408.5 376.3 492.2 467.0 391.9 123.3 Reduction: 20.8% 44.4% 40.9% 53.5%52.5% 42.6% 13.4%

EXAMPLE 14

Composition replicated Example 11 except disrupted cell ferment wasfiltered through a 30 kD molecular weight cutoff centrifuge tube toremove all proteins larger that 30 kD to demonstrate the functionalityof the low molecular weight peptides present in the disrupted cellferment.

EXAMPLE 15

Composition replicated Example 14 except disrupted cell ferment wasfiltered through a 10 kD molecular weight cutoff centrifuge tube toremove all proteins larger that 10 kD to demonstrate the functionalityof the low molecular weight peptides present in the disrupted cellferment.

TABLE 9 % By Weight Material Example 14 Example 15 D.C. Ferment <30 kD20.0% D.C. Ferment <10 kD 20.0% Water 72.5% 72.5% Ethoxylated LinearAlcohol - 7 mole ETO  7.5%  7.5% Adjust pH to 4.0 with Phosphoric AcidTOTAL 100.00%  100.00% 

Results showed no functional loss of metabolic efficiency due to removalof fermentation-produced compounds of greater that 30 kD molecularweight, or compounds greater than 10 kD in the disrupted cellfermentation product. Data infers there may be some larger molecularweight (>10 kD) compound(s) that may contribute an inhibitory effect,thereby reducing some degree of biological metabolic efficiency of theuncoupling factor of the present invention. Results were as follows:

TABLE 10 Efficacy of Low Molecular Weight Peptides Contained inDisrupted Cell Fermentation Product Nutrient TOC - mg/L Time ControlExample 11 Example 14 Example 15 Hour 0 863.0 866.9 865.7 861.4 2 779.1779.4 761.1 794.7 4 691.4 677.8 605.9 705.1 6 608.8 554.7 495.7 604.5 8499.7 401.9 279.4 268.5 ΔTOC 363.3 465.0 586.3 592.9 Reduction: 42.1%53.6% 67.7% 68.8%

EXAMPLE 16

The composition and process were identical to that of Example 1, exceptrefined LYCD utilizing dialysis membranes as set forth in U.S. Pat. No.5,356,874, yielding polypeptides having the molecular weights rangingbetween 6,000 and 17,000 daltons was introduced into the formula at alevel of 0.30%.

EXAMPLE 17

The composition and process are identical to that of Example 16, exceptrefined LYCD utilizing dialysis membranes as set forth in U.S. Pat. No.5,356,874, yielding polypeptides having the molecular weights rangingbetween 6,000 and 17,000 daltons replaced the fermentation product inthe formula at a level of 1.00%. Formulae for the two examples are asfollows:

TABLE 11 % By Weight Material Example 16 Example 17 Non-autolysedFerment 64.20% Refined LYCD 0.30% 1.00% Water 63.50% Ethoxylated LinearAlcohol - 7 mole ETO 26.60% 26.60% Alkyl Ether Sulfate, Sodium Salt(60%) 8.90% 8.90% Adjust pH to 4.0 with Phosphoric Acid Total 100.00%100.00%

Results of the carbon mass balance test using Examples 1, 16 and 17 at10 mg/l versus a negative control demonstrated that the addition of0.30% refined LYCD to the formula shown in Example 1, increased theuptake of carbon by 71% versus Example 1 formulation, and a carbonuptake increase of 230% when compared to the control. Respiration ofcarbon dioxide also increased by 83% versus the formula shown in Example1, and a seven-fold increase when compared to the control. When thefermentation product was completely replaced by 1.00% refined LYCD inExample 17, an 84% increase of carbon dioxide was observed when comparedto Example 1. Results were as follows:

TABLE 12 Carbon Mass Balance Comparisons of Modified AerobicFermentation Process Composition vs. Control Control Example 1 Example16 Example 17 Nutrient TOC/mg/l 0 Hour 891.2 901.3 889.5 899.1 4 Hours715.9 577.9 337.4 321.2 ΔTOC 175.3 323.4 552.1 577.9 Biomass TOC/mg/l 0Hour 81.2 53.3 76.9 96.9 4 Hours 224.0 195.4 360.4 401.0 ΔTOC 142.8142.1 283.5 304.1 Nutrient TOC to 81.5 43.9 51.3 52.6 Biomass TOC % CO2as Carbon 49.5 189.0 346.5 348.5 mg/l

The relative increase of biomass production for Examples 16 and 17 wasminor when compared to the significant increase of carbon uptake versusExample 1 and the Control. Additionally, the increase in biomass forExamples 16 and 17 was, in fact, directly proportional the increase ofCO2 production. Therefore, the shift in the percent of nutrient TOC tobiomass TOC (43.9% for Example 1 versus 51.3% for Example 16 and 52.6%for Example 17) may have reflected the margin of error inherent inconducting mass balance studies as opposed to determining theoreticalresults. An additional theory for the apparent upward shift in biomassproduction centers on the overabundance of assimilated carbon thatresulted in sufficient production of energy so as to begin to overcomethe uncoupling effect, thereby producing a slight increase in biomassproduction. Irrespective of the rationale for the shift observed, theuse of refined LYCD, in conjunction with surface-active agents, resultedin an uncoupling of biochemical pathways yielding in a significantincrease of carbon being metabolized and carbon dioxide respired,without the expected concomitant increase of biomass production.

EXAMPLE 18

This formulation replicates Example 3 except the fermentation productwas passed through a 10,000 dalton molecular weight cutoff centrifugetube to demonstrate the active proteins are of low molecular weightsize.

EXAMPLE 19

This formulation is designed to demonstrate that the observed metabolicactivity is not primarily a function of active enzymes. In the example,fermentation product was heated to 70 degrees C. for 24 hours,subsequently passed through a 10,000 dalton molecular weight cutoffcentrifuge tube. Formulae for the two examples were as follows:

TABLE 13 % By Weight Material Example 3 Example 18 Example 19 DisruptedCell Ferment 64.50% Disrupted Cell Ferment <10 kD 64.50% HeatedDisrupted Cell Ferment 64.50% <10 kD Ethoxylated Linear Alcohol - 26.60%26.60% 26.60% 7 mole ETO Alkyl Ether Sulfate, Sodium Salt 8.90% 8.90%8.90% (60%) Adjust pH to 4.0 with Phosphoric Acid Total 100.00% 100.00%100.00%

Results of the Mass Balance Bench Test using Examples 3, 18 and 19 at adose rate of 10 mg/l demonstrated a 22% decrease in the carbon uptakefor Example 18 relative to Example 3 when the protein fractions above10,000 daltons were removed from the fermentation product. The increaseof carbon uptake, however, was still substantially higher compared tothe control (by 103%). Further, the amount of biomass formed withExample 18 was 20% lower, keeping the ratio of nutrient consumed tobiomass produced essentially the same as in Example 3. Heating of thedisrupted cell fermentation product prior to removal of the proteinfractions greater than 10,000 daltons had little effect on the carbonmass balance. Test results were as follows:

TABLE 14 Mass Balance Comparisons of Modified Aerobic FermentationProcess Compositions and Yeast-Derived Low Molecular Weight Proteins vs.Control Control Example 3 Example 18 Example 19 Nutrient TOC/mg/l 0 Hour891.2 854.0 922.8 899.8 4 Hours 715.9 398.1 566.1 552.8 ΔTOC 175.3 455.9356.7 347.0 Biomass TOC/mg/l 0 Hour 81.2 71.7 71.0 36.3 4 Hours 224.0321.9 271.6 220.5 ΔTOC 142.8 250.2 200.6 184.2 % Nutrient TOC to 81.554.9 56.2 53.1 Biomass TOC % Carbon Dioxide as 49.5 252.0 211.5 157.5Carbon mg/l

A feature of the present invention is the ability to reduce the amountof aeration required in aerobic wastewater processes, which would resultin energy savings; normally a substantial expense for wastewatertreatment. This feature is possible with the present invention due toincreased metabolism of BOD. In the early stages of the process, oxygendemand is greater for the treated test cylinder versus the control.Since a greater uptake of BOD is realized earlier in the process, therespiration of bacteria will decline in the absence of available orsufficient biologically available nutrient. Therefore, the oxygen demanddecreases sooner for the treated test cylinder, thus resulting in asurplus of oxygen at the conclusion of the test period. This wouldpermit a reduction in the total amount of aeration applied to theprocess. Results of that study were as follows:

TABLE 15 Relationship of Carbon Metabolism, Dissolved Oxygen and pH forExample 3 and Control Nutrient TOC- Dissolved Oxygen- mg/L pH mg/L Con-Exam- Con- Exam- Con- Exam- Time trol ple 3 trol ple 3 trol ple 3 Hour 0980.5 980.5 5.94 5.94 0.43 0.43 2 924.3 908.0 5.97 6.06 1.16 2.98 4937.6 855.4 6.31 6.49 0.15 0.14 6 792.8 638.6 6.35 6.85 0.16 0.14 8743.0 424.8 6.44 7.35 0.22 0.14 10 581.6 447.0 6.86 7.27 0.21 3.84 12502.4 451.4 7.42 7.69 0.18 5.71 14 451.4 482.5 7.16 7.44 0.11 4.36 16465.5 404.8 7.73 7.43 0.26 5.93

Results shown in Table 15 demonstrate the relationship between the levelof bio-available nutrient (BOD) and the level of unutilized dissolvedoxygen under comparable fixed, environmental and operating conditionsfor control and treated samples. The D.O of the treated sample increasedafter 8 hours dwell time when the bio-available nutrient declined, inthis exhibit, below 450 mg/l of carbon. The D.O. of the control sampleremained below 0.3 mg/l after 16 hours since the organic carbon levelremained above 450 mg/l.

EXAMPLE 20

The aerobic fermentation process is not conducted under sterileconditions. Therefore, significant levels of bacterial contaminationexist at the conclusion of the fermentation process. The presence ofthis contamination can facilitate a rapid degradation of the activepeptides and surface-active agents, thereby rendering the compositionless effective for its intended application. Therefore, the use of abroad-spectrum antimicrobial system is deemed useful. This example,based on the composition used in Example 3, illustrates a stabilizationsystem that provides antimicrobial activity against a broad spectrum oforganisms produced during the yeast fermentation process. Thecomposition contains about 3 times the level required to adequatelypreserve the composition, thus providing a composition capable of beingdiluted with about 2 parts of water.

EXAMPLE 21

The treatment of large municipal wastewater treatment facilities isaccomplished by utilizing a treatment level of about 1 to about 3 mg/lof Example 20. However, treatment of small wastewater flows can bedifficult to accurately meter at this low dose rate. Therefore, it isdesirable to provide a more dilute version of Example 20 wherein thestabilizer/preservative system is present at sufficient levels so as toprevent premature degradation of the composition. Example 21 is preparedwith 35.5% of the active components used in Example 20 and the balanceof the composition is comprised of water.

TABLE 16 % By Weight Material Example 3 Example 20 Example 21 DisruptedCell Ferment 64.50% 54.60% 19.38% Ethoxylated Linear 26.60% 22.50% 7.50%Alcohol - 7 mole ETO Alkyl Ether Sulfate, 8.90% 7.50% 2.50% Sodium Salt(60%) Propylene Glycol 14.60% 5.18% Sodium Benzoate 0.35% 0.10% MethylParaben 0.35% 0.10% Propyl Paraben 0.10% 0.03% Water 65.21% Adjust pH to4.0 with Phosphoric Acid Total 100.00% 100.00% 100.00%Examples 20 and 21 both demonstrated the ability to control both yeastand bacterial residual contamination resulting from the fermentationprocess.

All patents and literature references cited in this specification arehereby incorporated by reference in their entirety.

Thus, the compounds, systems and methods of the present inventionprovide many benefits over the prior art. While the above descriptioncontains many specificities, these should not be construed aslimitations on the scope of the invention, but rather as anexemplification of the preferred embodiments thereof. Many othervariations are possible.

Accordingly, the scope of the present invention should be determined notby the embodiments illustrated above, but by the appended claims andtheir legal equivalents.

What is claimed is:
 1. A method for accelerating nutrient uptake inbacteria without a commensurate increase of biomass, comprisingcontacting said bacteria with a mixture of an aerobic yeast fermentationsupernatant and a surface-active agent selected from the groupconsisting of a nonionic surfactant, a combination of nonionic andanionic surfactants, an ethoxylated linear alcohol, and an alkyl ethersulfate, whereby the nutrient uptake in said bacteria is increasedwithout a commensurate increase of biomass, wherein the mixture of theaerobic yeast fermentation supernatant and the surface-active agent isobtained by: fermenting under aerobic conditions a plurality of yeastcells in the presence of a nutrient source, wherein the yeast cells areselected from a group consisting of Saccharomyces cerevisiae,Kluyveromyces marxianus, Kluyveromyces lactis, Candida utilis,Zygosaccharomyces, Pichia, and Hansanula , after the fermenting step,subjecting the yeast cells to heat stress by increasing the fermentationtemperature to between 40° C. and 60° C. for 2 to 24 hours to obtain afermentation product, centrifuging the fermentation product to obtainthe aerobic fermentation supernatant containing peptides, and combiningthe aerobic fermentation supernatant with the surface-active agent. 2.The method of claim 1, further comprising disrupting the cellularstructure of the plurality of yeast cells subsequent to the heating. 3.The method of claim 2, wherein said disrupting the cellular structure ofthe plurality of yeast cells releases intracellular peptides from theyeast cells into the aerobic fermentation supernatant.
 4. The method ofclaim 1, wherein the nutrient source comprises a sugar.
 5. The method ofclaim 4, wherein the nutrient source further comprises one or more ofdiastatic malt, diammonium phosphate, magnesium sulfate, ammoniumsulfate zinc sulfate, and ammonia.
 6. The method of claim 2, whereinsaid disrupting the cellular structure of the plurality of yeast cellscomprises physically disrupting or chemically disrupting the cellularstructure of the plurality of yeast cells.
 7. The method of claim 6,wherein said physically disrupting comprises subjecting the yeast cellsto one or more of a French Press, a ball mill, or a high-pressurehomogenizer.
 8. The method of claim 6, wherein said chemicallydisrupting comprises combining said plurality of yeast cells with asecond surface-active agent.
 9. The method of claim 6, wherein saidchemically disrupting comprises adding about 2.5% to about 10% of asurfactant to a yeast cell suspension and agitating the mixture at atemperature of about 25° C. to about 35° C.
 10. The method of claim 6,wherein said disrupting comprises both physically disrupting andchemically disrupting a plurality of said yeast cells.
 11. The method ofclaim 1, further comprising cooling the fermentation temperature to lessthan 25° C. subsequent to the heating step.
 12. The method of claim 1,wherein said bacteria are mixed in with wastewater.
 13. The method ofclaim 1, wherein said bacteria are present in a sewage collectionsystem.
 14. The method of claim 13, wherein said sewage collectionsystem comprises a system selected from a cross-flow membrane filtrationsystem or a cooling tower.
 15. A method for accelerating nutrient uptakein bacteria or yeast without a commensurate increase in biofilmproduction, comprising contacting said bacteria or yeast with a mixtureof an aerobic yeast fermentation supernatant and a surface-active agentselected from the group consisting of a nonionic surfactant, acombination of nonionic and anionic surfactants, an ethoxylated linearalcohol, and an alkyl ether sulfate, whereby the nutrient uptake in saidbacteria or yeast is increased without a commensurate increase inbiofilm production, wherein the mixture of the aerobic yeastfermentation supernatant and the surface-active agent is obtained by:fermenting under aerobic conditions a plurality of yeast cells in thepresence of a nutrient source, wherein the yeast cells are selected froma group consisting of Saccharomyces cerevisiae, Kluyveromyces marxianus,Kluyveromyces lactis, Candida utilis, Zygosaccharomyces, Pichia, andHansanula, subjecting the yeast cells to heat stress by increasing thefermentation temperature to between 40° C. and 60° C. for 2 to 24 hours,centrifuging the fermentation product to obtain the aerobic fermentationsupernatant containing peptides, and combining the aerobic fermentationsupernatant with the surface-active agent.
 16. The method of claim 15,further comprising cooling the fermentation temperature to less than 25°C. subsequent to the heating step.